PROJECT SUMMARY Cytotoxic T lymphocytes (CTLs) eliminate target cells primarily through polarized secretion of the contents of membrane-enclosed cytolytic granules, specialized secretory lysosomes loaded with the pore-forming protein perforin and the serine proteases granzymes. When a CTL recognizes its target, the contact area between the CTL and the target cell forms a highly organized and stable structure known as the immunological synapse. Subsequently, cytolytic granules migrate from their dispersed locations in the cytosol toward the immunological synapse, where they dock and fuse with the plasma membrane to secrete their contents. Released cytolytic molecules then enter the target cell and initiate programmed cell death (apoptosis). The regulation of cytolytic granule secretion is still poorly understood at the molecular level. The major goal of this pilot project is to dissect cytolytic granule secretion in CTLs using unbiased genome-wide CRISPR-Cas9 genetic screens. In our preliminary studies, we developed novel platforms to genetically dissect mammalian vesicle transport pathways using genome-wide CRISPR screens. Furthermore, we developed assays to measure cytolytic granule secretion in CTLs. Here, we will take strategic advantage of these systems to dissect cytolytic granule secretion using an unbiased CRISPR genetic screen. We will then validate the identified genes using a pooled secondary screen as well as individual gene editing. Finally, we will characterize the knockout phenotype of selected candidate genes in CTLs. If successfully accomplished, this proposed research will provide the first genome-scale view of cytolytic granule secretion, and will substantially expand our knowledge of how CTLs destroy target cells. Insights gleaned from these studies will help improve the efficacy and safety of cancer immunotherapy, and will facilitate the development of new therapeutics for immune disorders caused by defective cytolytic granule secretion.